Human growth hormone (hGH), also known as somatropin (INN) or somatotropin, is a protein hormone produced and secreted by the somatotropic cells of the anterior pituitary. Human growth hormone plays a key role in somatic growth in childhood and in metabolism in adulthood through its effects on the metabolism of proteins, carbohydrates and lipids.
Human growth hormone is a single polypeptide chain of 191 amino acids (Bewly et al, 1972) having two disulfide bonds, one between Cys-53 and Cys-165, forming a large loop in the molecule, and the other between Cys-182 and Cys-189, forming a small loop near the C-terminus. The DNA sequence that confirmed the amino acid sequence was reported by Martial et al (1979). Purified hGH is a white amorphous powder in its lyophilized form. It is readily soluble (concentrations>10 mg/L) in aqueous buffers at pH in a range of 6.5 to 8.5.
In solution, hGH exists predominantly as a monomer, with a small fraction as dimers and higher molecular weight oligomers. Under certain conditions, hGH can be induced to form larger amounts of dimers, trimers and higher oligomers.
Several derivatives of hGH are known, including naturally-occurring derivatives, variants and metabolic products, degradation products primarily of biosynthetic hGH and engineered derivatives of hGH produced by genetic methods. One example of a naturally-occurring derivative of hGH is GH-V, a variant of growth hormone found in the placenta. Other members of the gene locus are described in Chen et al (1989).
Methionyl hGH was the first form of hGH to be produced through recombinant DNA technology. This compound is actually a derivative of hGH having one additional methionine residue at its N-terminus (Goeddel et al, 1979).
A naturally-occurring variant of hGH called 20-K-hGH has been reported to occur in the pituitary as well as in the bloodstream (Lewis et al, 1978; Lewis et al, 1980). This compound, which lacks the 15 amino acid residues from Glu-32 to Gln-46, arises from an alternative splicing of the messenger ribonucleic acid (DeNoto et al, 1981). This compound shares many, but not all of the biological properties of hGH.
20-K-hGH is made in the pituitary and secreted into the blood. It makes up about 5% of growth hormone output of adults, and about 20% of growth hormone output of children. It has the same growth promoting activity as 22 kD growth hormone, and has been reported to have equal to or greater the amount of lipolytic activity as the 22 kD form. It binds to growth hormone receptors with equal affinity as the 22 kD growth hormone, and has one tenth the lactogenic (prolactin-like) bioactivity as the 22 kD hormone. Unlike 22 kD, the 20-k-hGH has weak anti-insulin activity.
A number of derivatives of hGH arise from proteolytic modifications of the molecule. The primary pathway for the metabolism of hGH involves proteolysis. The region of hGH around residues 130-150 is extremely susceptible to proteolysis, and several derivatives of hGH having nicks or deletions in this region have been described (Thorlacius-Ussing, 1987). This region is in the large loop of hGH, and cleavage of a peptide bond there results in the generation of two chains that are connected through the disulfide bond at Cys-53 and Cys-165. Many of these two-chain forms are reported to have increased biological activity (Singh et al, 1974). Many derivatives of human growth hormone have been generated artificially through the use of enzymes. The enzymes trypsin and subtilisin, as well as others, have been used to modify hGH at various points throughout the molecule (Lewis et al, 1977; Graff et al, 1982). One such derivative, called two-chain anabolic protein (2-CAP), was formed through the controlled proteolysis of hGH using trypsin (Becker et al, 1989). 2-CAP was found to have biological properties very distinct from those of the intact hGH molecule, in that the growth-promoting activity of hGH was largely retained and most of the effects on carbohydrate metabolism were abolished.
Asparagine and glutamine residues in proteins are susceptible to deamidation reactions under appropriate conditions. Pituitary hGH has been shown to undergo this type of reaction, resulting in conversion of Asn-152 to aspartic acid and also, to a lesser extent, conversion of Gln-137 to glutamic acid (Lewis et al, 1981). Deamidated hGH has been shown to have an altered susceptibility to proteolysis with the enzyme subtilisin, suggesting that deamidation may have physiological significance in directing proteolytic cleavage of hGH. Biosynthetic hGH is known to degrade under certain storage conditions, resulting in deamidation at a different asparagine (Asn-149). This is the primary site of deamidation, but deamidation at Asn-152 is also seen (Becker et al, 1988). Deamidation at Gln-137 has not been reported in biosynthetic hGH.
Methionine residues in proteins are susceptible to oxidation, primarily to the sulfoxide. Both pituitary-derived and biosynthetic hGH undergo sulfoxidations at Met-14 and Met-125 (Becker et al, 1988). Oxidation at Met-170 has also been reported in pituitary but not biosynthetic hGH. Both desamide hGH and Met-14 sulfoxide hGH have been found to exhibit full biological activity (Becker et al, 1988).
Truncated forms of hGH have been produced, either through the actions of enzymes or by genetic methods. 2-CAP, generated by the controlled actions of trypsin, has the first eight residues at the N-terminus of hGH removed. Other truncated versions of hGH have been produced by modifying the gene prior to expression in a suitable host. The first 13 residues have been removed to yield a derivative having distinctive biological properties (Gertler et al, 1986) in which the polypeptide chain is not cleaved.
Although human growth hormone was originally obtained from pituitary glands of cadavers, these preparations were not electrophoretically homogeneous, and antibodies appeared in the serum of patients treated with preparations of the order of 50% purity, the immunogenicity being attributed to inactive components. Recombinant DNA technology permitted production of an unlimited supply of hGH in a number of different systems. Purification of hGH from the culture medium is facilitated by the presence of only low amounts of contaminating proteins. In fact, it has been shown that hGH can be purified on a laboratory scale by a single purification step on a reversed-phase HPLC column (Hsiung et al (1989).
Recombinant human growth hormone, rhGH, is produced by Serono International S.A. as SEROSTIM®, which product has been given accelerated FDA approval for treating weight loss and wasting in AIDS patients. SAIZEN® is recombinant human growth hormone indicated for GH deficiency in children, for Turner syndrome in girls, as well as chronic renal failure in children. PROTROPIN®, produced by Genentech, Inc. (South San Francisco, Calif.), differs slightly in structure from natural sequence hGH, having an additional methionine residue at the N-terminus. Recombinant hGH is generally marketed as vials containing hGH plus additional excipients, e.g., glycine and mannitol, in a lyophilized form. A companion diluent vial is provided, allowing the patient to reconstitute the product to the desired concentration prior to administration of the dose. Recombinant hGH can also be marketed in other well-known manners, such as pre-filled syringes.
In order for hGH to be available commercially as a therapeutic, stable formulations must be prepared. Such formulations must be capable of maintaining activity for appropriate storage times and be acceptable for administration by patients.
Human GH has been formulated in a variety of ways. By way of example, U.S. Pat. No. 5,096,885 discloses a stable pharmaceutically acceptable formulation of hGH comprising, in addition to the hGH, glycine, mannitol, a buffer and optionally a non-ionic surfactant, the molar ratio of hGH:glycine being 1:50.
WO 93/19776 discloses injectable formulations of GH comprising citrate as buffer substance and optionally growth factors such as insulin-like growth factors or epidermal growth factor, amino acids such as glycine or alanine, mannitol or other sugar alcohols, glycerol and/or a preservative such as benzyl alcohol.
WO 94/101398 discloses a GH formulation containing hGH, a buffer, a non-ionic surfactant and, optionally, mannitol, a neutral salt and/or a preservative.
EP-0131864 describes an aqueous solution of proteins with molecular weight above 8500 daltons, which have been protected from adsorption at interfaces, against denaturing and against precipitation of the protein by addition of a linear polyoxyalkylene chain-containing surface-active substance as a stabilising agent.
EP-0211601 discloses a growth promoting formulation comprising an aqueous mixture of growth promoting hormone and a block copolymer containing polyoxyethylene-polyoxypropylene units and having an average molecular weight of about 1,100 to about 40,000 which maintains the fluidity of the growth promoting hormone and its biological activity upon administration.
WO 97/29767 discloses a liquid formulation comprising a growth hormone, trisodium citrate dihydrate, sodium chloride, sodium hydroxide, benzyl alcohol, Pluronic F-68, said formulation having a pH of 5.6.
U.S. Pat. No. 5,567,677 discloses liquid formulations comprising human growth hormone, sodium citrate, sodium phosphate, glycine, mannitol, optionally benzyl alcohol.
Pharmaceutical preparations of hGH tend to be unstable, particularly in solution. Chemically degraded species such as deamidated or sulfoxylated forms of hGH occur, and dimeric or higher molecular weight aggregated species may result from physical instability (Becker et al (1988); Becker et al., 1987; Pearlman and Nguyen (1989)).
As a consequence of the instability of hGH in solution, pharmaceutical formulations of hGH are generally in lyophilised form, which must then be reconstituted prior to use. Reconstitution is usually carried out by the addition of a pharmaceutically acceptable diluent such as sterile water for injection, sterile physiological saline or an appropriate sterile physiologically acceptable diluent.
Reconstituted solutions of hGH are preferably stored at 4° C. to minimise chemical and physical degradation reactions, however some degradation will occur during such storage which can be for a period of up to 14 days.
A pharmaceutical formulation of hGH provided in a liquid form, particularly one that maintains stability of hGH without formation of precipitation or aggregation or any other particulate matter over a prolonged period of time, would be particularly advantageous.
Therefore, it is an object of the present invention to provide liquid formulations of growth hormone that do not result in the formation of undesirable particulate matter and that has a prolonged storage time.